Hantaviruses (HVs) predominantly infect the endothelial cell (EC) lining of capillaries and nonlytically cause vascular leakage. Andes virus (ANDV) and Sin Nombre virus (SNV) infect virtually all pulmonary ECs(109) and cause 35% fatal acute pulmonary edema (HV pulmonary syndrome-HPS). However, ANDV is the only HV spread person to person and the only HV that causes lethal HPS-like disease in hamsters. Pathogenic HVs regulate early innate immune responses in order to successfully replicate in ECs and we recently revealed that ANDV contains an additional interferon (IFN) regulating determinant that explains unique ANDV spread and virulence and is a potential target for attenuating ANDV. HVs are inhibited by IFN added prior to or early after infection, and in order to replicate in human ECs pathogenic HVs regulate early IFN induction. Nonpathogenic PHV, fails to regulate IFN induction or replicate in human ECs, and failed IFN regulation explains why PHV is not a human pathogen. Except for PHV, HV Gn proteins contain cytoplasmic tail (GnT) elements that inhibit RIG-I/TBK1 directed IRF3 phosphorylation and IFN? induction. In contrast, N proteins from TULV, SNV, NY-1V, HTNV and PHV fail to inhibit RIG-I/TBK1 directed IFN induction. Unpredictably, the ANDV N protein inhibited RIGI/TBK1 directed IFN induction. In contrast, N protein from a nonpathogenic S. American HV, MAPV, failed to inhibit IFN signaling, yet MAPV N differs by only 12 dissimilar residues from ANDV N. ANDV N protein inhibits TBK1 autophosphorylation that is required for TBK1 phosphorylation of IRF3 and our recent proteomics screen found that ANDV N binds a single IFN regulating protein in ECs, TRIM21. Reciprocal TRIM21 IPs validated ANDV N, but not TULV N, as a TRIM21 binding partner. Preliminary data indicates that mutating ANDV residues to MAPV N residues prevents IFN regulation and TRIM21 binding, and provides a mechanism for attenuating ANDV. Our findings suggest that IFN regulation by the ANDV N protein is a novel virulence determinant. This may explain why ANDV uniquely causes viremia and lethal HPS disease in Syrian hamsters and why ANDV is the only HV spread from person to person. These findings permit us to propose defining the role of N and Gn directed IFN regulation in attenuating ANDV and preventing lethal HPS in Syrian hamsters. We define mechanisms by which N and Gn proteins inhibit RIG-I/TBK1 signaling pathways and identify mutations that abolish regulation. We evaluate ANDV reassortants and mutants for attenuation in a lethal Syrian hamster HPS disease model. We define HV virulence determinants by identifying N/Gn residues of ANDV, MAPV & SNV required to regulate RIG-I/TBK1/IRF3 signaling pathways. We analyze ANDV regulation of IFN inductionin ECs, ANDV N regulation of TRIM21, and N/Gn synergy in IFN regulation. ANDV/MAPV reassortants and N/Gn-?IFN rANDV mutants are evaluated for their attenuation and ability to protect Syrian hamsters from Wt ANDV infection.